1. Field of the Invention
This invention relates generally to the field of detecting hazardous chemicals and chemical analysis, to include portable automatic sensing devices. More particularly, the present invention relates to methods, compositions, devices and kits thereof useful in the detection of chemical agents, insecticides and other acetylcholinesterase (AChE) inhibitors, modifiers and ligands.
2. Background Information
Acetylcholinesterase (AChE), a serine hydrolase in the α/β-fold hydrolase protein superfamily, terminates nerve signals by catalyzing hydrolysis of the neurotransmitter acetylcholinesterase at a diffusion-limited rate. A number of nerve toxins, including insecticides and organophosphates, act through binding to and inhibiting AChE.
Organophosphorus and organosulfur compounds, are used extensively in insecticides and are highly toxic to many organisms including humans. Insecticide residues are found in soil and groundwater, and the detection of these residues is important for their elimination from the environment and to protect the health of both humans and animals. Organophosphorus compounds are also used in nerve agents, such as sarin, phosphine, soman, and tabun, for chemical warfare purposes.
These agents are some of the most potent toxic agents and are specific inhibitors of acetylcholinesterase (AChE).
Acetylcholine is an essential neurotransmitter that affects parasympathetic synapses (autonomic and CNS), sympathetic preganglionic synapses, and the neuromuscular junction (see, e.g., Taylor et al., in Basic Neurochemistry, 5th ed., 1993, (Siegal et al., eds.), Chapter 11, pp. 231-260, Raven Press, New York, N.Y.). Hydrolysis of acetylcholine by acetylcholinesterase, present in nervous tissue, normally limits the duration of action function. Organophosphate (e.g., Malathion, Parathion, Diasinon, Dursban) and carbamate (e.g., Sevin, Furadan) insecticides exert their toxicity by inhibiting the action of acetylcholinesterase and thereby causing a pronounced cholinergic response (Arron et al., Insecticides: Organophosphate and Carbamates in Goldfrank's Toxicologic Emergencies, 1994, (Goldfrank et al., eds.), Appleton & Lange, Norwalk, Conn.). Enzyme inhibition is the consequence of phosphorylation (organophosphates) or carbamylation (carbamates) of the cholinesterase-active site serine residue. The resulting phosphoroyl-serine bond is stable; therefore, enzyme inhibition is physiologically irreversible, whereas the carbamyl-serine bond undergoes spontaneous hydrolysis with regeneration of enzyme activity (24-48 h). For this reason and because of poor CNS penetration, carbamate insecticide neurotoxicity is less severe and of shorter duration than that for the organophosphates (Tietz Textbook of Clinical Chemistry, 1999, (Burtis et al., eds.), W. B. Saunders Company, Philadelphia, Pa.).
Excess synaptic acetylcholine stimulates muscarinic receptors (peripheral and CNS) and stimulates but then depresses and paralyzes nicotinic receptors. The CNS neurotoxic effects include restlessness, agitation, lethargy, confusion, slurred speech, seizures, coma, cardiorespiratory depression, or death.
The need for the reliable determination of these cholinesterase inhibitors has led to the development of a number of sophisticated instrumental methods, mostly involving the use of gas and liquid chromatography and mass spectrometry. Also a number of liquid phase chemiluminescence procedures have been developed for the determination of inorganic and organic species mostly utilizing the luminol and peroxyoxalate reactions. See Robards K. and Worsfold P. J., Anal Chem Acta (1992) 266:147.
These traditional methods are not practical for individual use as the methods are time consuming and complicated and the instruments utilized are expensive, non-portable and require high maintenance. Additionally, the measurement of nerve agents in mixtures with these traditional methods requires cumbersome extraction and manipulation procedures.
Thus, biosensors were developed as an alternative to the traditional gas and liquid chromatography and mass spectrometry technology. Generally, biosensors include those which are enzyme-based and bioaffinity-based. An enzymatic biosensor uses an enzymatic or metabolic process to detect a reaction product which occurs between an incoming substrate and an immobilized enzyme. A bioaffinity sensor relies on a biological binding event of a target substance.
Many existing methods for the detection of organophosphates and cumulative inhibition of cholinesterases lack sensitivity since they are based on inhibition of basal activities rather than accumulation of the inhibitory conjugate. Basal activities vary substantially between subjects resulting in inconsistency in present assays.
Existing monitoring methods routinely require expensive laboratory procedures involving sample transport or preparations of samples for assay.
Rapid analysis of toxic materials in the areas of food and water analysis, environmental monitoring, and in industrial settings is a problem that continues to exist and is currently addressed by time-consuming, expensive methods or by techniques that may be described as inadequate.
Many problems associated with exposure to toxic materials could be avoided or minimized by a detection procedure which gives near “real-time” indication of the presence of toxic gases. Equally important are the characteristics of economy, small size, and ease of use for the successful application of such devices.
Accordingly, there is a need for a method of detecting, quantifying, and evaluating hazards which provides for early detection and which can detect low levels of toxic materials. The present invention satisfies this need, as well as others.